Germline or somatic GPR101 duplication leads to X-linked acrogigantism: a clinico-pathological and genetic study

Abstract

Non-syndromic pituitary gigantism can result from AIP mutations or the recently identified Xq26.3 microduplication causing X-linked acrogigantism (XLAG). Within Xq26.3, GPR101 is believed to be the causative gene, and the c.924G > C (p.E308D) variant in this orphan G protein-coupled receptor has been suggested to play a role in the pathogenesis of acromegaly.We studied 153 patients (58 females and 95 males) with pituitary gigantism. AIP mutation-negative cases were screened for GPR101 duplication through copy number variation droplet digital PCR and high-density aCGH. The genetic, clinical and histopathological features of XLAG patients were studied in detail. 395 peripheral blood and 193 pituitary tumor DNA samples from acromegaly patients were tested for GPR101 variants.We identified 12 patients (10 females and 2 males; 7.8 %) with XLAG. In one subject, the duplicated region only contained GPR101, but not the other three genes in found to be duplicated in the previously reported patients, defining a new smallest region of overlap of duplications. While females presented with germline mutations, the two male patients harbored the mutation in a mosaic state. Nine patients had pituitary adenomas, while three had hyperplasia. The comparison of the features of XLAG, AIP-positive and GPR101&AIP-negative patients revealed significant differences in sex distribution, age at onset, height, prolactin co-secretion and histological features. The pathological features of XLAG-related adenomas were remarkably similar. These tumors had a sinusoidal and lobular architecture. Sparsely and densely granulated somatotrophs were admixed with lactotrophs; follicle-like structures and calcifications were commonly observed. Patients with sporadic of familial acromegaly did not have an increased prevalence of the c.924G > C (p.E308D) GPR101 variant compared to public databases.In conclusion, XLAG can result from germline or somatic duplication of GPR101. Duplication of GPR101 alone is sufficient for the development of XLAG, implicating it as the causative gene within the Xq26.3 region. The pathological features of XLAG-associated pituitary adenomas are typical and, together with the clinical phenotype, should prompt genetic testing.

Trial registration: ClinicalTrials.gov NCT00461188.

Keywords: CNV mutation; GPR101; Gigantism; Pituitary; XLAG.

Fig. 1

Genomic rearrangements identified in eight subjects with duplications encompassing GPR101. The HD-aCGH log₂ ratio plot for each rearrangement is aligned within the genomic interval of ChrX: 135379766–136479766 (x axis), with the identification of each subject on the y axis. The cluster of red dots shows interrogating oligonucleotide probes that demonstrate increased hybridization intensity, revealing a gain in copy number. The gene content included in the duplicated region is shown underneath the aligned HD-aCGH log₂ ratio plots. The symbols on the right side of the gene names represent the structure of the genes, with vertical lines representing exons. GPR101, a single exon gene, is highlighted in red. The region between the black vertical lines across the log₂ ratio plots and gene track represent the smallest region of overlap encompassing solely GPR101, but not the other three genes (CD40LG, ARHGEF6 and RBMX) in the smallest region of overlap of previously published patients. Red probes, log₂ ratio > 0.25; black probes, −0.25 ≤ log₂ ratio ≤ 0.25; green probes, log₂ ratio < −0.25

Fig. 2

Adenomas occurring in XLAG patients are characterized by distinct populations of acidophilic and chromophobic cells (a HE - x10; b HE x40); staining for reticulin fibers highlights the lobular and cordonal architecture of XLAG-related adenomas (c Gordon-Sweet’s silver impregnation - x10); perivascular connective tissue containing thickened and distorted reticulin fibers (d Gordon-Sweet’s silver impregnation – x40)

Fig. 3

Secondary features of XLAG adenomas include pseudo-follicles containing colloid-like material (asterisk) (a HE – x20), isolated cells with large, irregular nucleus (b HE – x20), and scattered calcifications (arrow) (c HE – x20)

Fig. 4

DG somatotrophs represent the predominant component in XLAG adenomas (a GH staining with immunoperoxidase – x10), while neoplastic lactotrophs appear as smaller areas (b PRL staining, immunoperoxidase – x10); some tumor cells express the common α-subunit (c immunoperoxidase – x20); double immunofluorescence for GH (green) and PRL (red) shows lack of co-localization of the two hormones in neoplastic cells (d immunofluorescence – x63)

Fig. 5

Immunostaining demonstrates areas composed of cells with faint GH expression containing fibrous bodies (arrow) (a immunoperoxidase – x20; insert – x40). Fibrous bodies are positive for cytokeratin CAM5.2 (b immunoperoxidase – x20); the transcription factor PIT-1 is expressed in the majority of the cells (c immunoperoxidase – x20); the Ki-67 labelling index is <3 % in all cases (d immunoperoxidase – x20)